Loss of sphingosine 1-phosphate receptor 3 gene function impairs injury-induced stromal angiogenesis in mouse cornea

• Published in: Laboratory investigation Vol. 101; no. 2; pp. 245 - 257
• Main Authors: Yasuda, Shingo, Sumioka, Takayoshi, Iwanishi, Hiroki, Okada, Yuka, Miyajima, Masayasu, Ichikawa, Kana, Reinach, Peter S., Saika, Shizuya
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2021; Nature Publishing Group; Nature Publishing Group US
• Subjects: Angiogenesis; Animals; Blood vessels; Cadherins; Cells, Cultured; Cornea; Cornea - cytology; Cornea - metabolism; Corneal Injuries - metabolism; Endothelial cells; Epithelial cells; Fibroblasts; Gene expression; Growth factors; Human Umbilical Vein Endothelial Cells; Humans; Injuries; Kinases; Macrophages; Mice; Mice, Knockout; Microvasculature; Neovascularization, Physiologic - drug effects; Neovascularization, Physiologic - genetics; Phosphorylation; Receptors; Signal transduction; Signal Transduction - drug effects; Signal Transduction - genetics; Signaling; Sphingosine 1-phosphate; Sphingosine kinase; Sphingosine-1-Phosphate Receptors - antagonists & inhibitors; Sphingosine-1-Phosphate Receptors - genetics; Sphingosine-1-Phosphate Receptors - metabolism; Thiazolidines - pharmacology; Transforming growth factor-b1; Vascular endothelial growth factor; Vascularization
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1038/s41374-020-00505-1

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated through sphingosine kinase1 (SPK1)-mediated phosphorylation of sphingosine. We show here that injury-induced S1P upregulation increases corneal neovascularization through stimulating S1PR3, a cognate receptor. since this response was suppressed in S1PR3-knockout mice. Furthermore, Cayman10444, a selective S1PR3 inhibitor, reduced this response in WT mice. Such reductions in neovascularization were associated with reduced vascular endothelial growth factor A (VEGF-A) mRNA expression levels in WT TKE2 corneal epithelial cells and macrophages treated with CAY10444 as well as macrophages isolated from S1PR3 KO mice. S1P increased tube-like vessel formation in human vascular endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMECs) cells expressing S1PR3. In S1PR3 KO mice, TGFβ1-induced increases in αSMA gene expression levels were suppressed relative to those in the WT counterparts. In S1PR3 deficient macrophages, VEGF-A expression levels were lower than in WT macrophages. Transforming growth factor β1(TGFβ1) upregulated SPK1 expression levels in ocular fibroblasts and TKE2 corneal epithelial cells. CAY10444 blocked S1P-induced increases in VEGF-A mRNA expression levels in TKE2 corneal epithelial cells. Endogenous S1P signaling upregulated VEGF-A and VE-cadherin mRNA expression levels in HUVEC. Unlike in TKE2 cells, SIS3 failed to block TGFβ1-induced VEGF-A upregulation in ocular fibroblasts. Taken together, these results indicate that injury-induced TGFβ1 upregulation increases S1P generation through increases in SPK1 activity. The rise in S1P formation stimulates the S1PR3-linked signaling pathway, which in turn increases VEGF-A expression levels and angiogenesis in mouse corneas. Injury-induced transforming growth factor β1 increases sphingosine 1-phosphate (S1P) generation by upregulating in sphingosine kinase 1 activity. The high levels of S1P formation stimulate the S1PR3-linked signaling pathway, which in turn increases vascular endothelial growth factor-A expression levels and angiogenesis in mouse corneas.